The various tissues of cereal grains have diverse functions during grain development, dormancy and after germination.
For example, the pericarp and seed coat tissues are concerned with the protection of the seed during development and during dormancy. However, by grain maturity, these outer grain tissues have died and the tissue residues consist almost entirely of cell wall residues. The nuclear tissue between the seed coat and the aleurone surface is involved in transfer of nutrients to the developing grain, however, at maturity, this tissue has also collapsed to leave cell wall remnants. The thin walled cells of the starchy endosperm of mature grain are dead, but are packed with starch and storage protein. In contrast, the thick-walled, nucleated, aleurone cells are alive at grain maturity, and are packed with protein bodies and lipid droplets. At the interface of the starchy endosperm lies the scutellum, which functions in delivering nutrients to the developing endosperm and, during germination, transfers digestion products of the endosperm reserves to the developing embryo.
The different structure and function of each tissue type in the grain are determined, at least in part, by the cell wall composition of each of these cell types.
Non-cellulosic polysaccharides are key components in the cell walls of cereal grain tissues and include, for example, (1,3;1,4)-β-D-glucans, heteroxylans (mainly arabinoxylans), glucomannans, xyloglucans, pectic polysaccharides and callose. These non-cellulosic polysaccharides usually constitute less than 10% of the overall weight of the grain, but nevertheless are key determinants of grain quality.
Although the precise physical relationships between individual non-cellulosic polysaccharides and other wall components have not been described, it is generally considered that in the wall, microfibrils of cellulose are embedded in a matrix phase of non-cellulosic polysaccharides and protein. Wall integrity is maintained predominantly through extensive non-covalent interactions, especially hydrogen bonding, between the matrix phase and microfibrillar constituents. In the walls of some grain tissues covalent associations between heteroxylans, lignin and proteins are present. The extent of covalent associations between components also varies with the wall type and genotype.
Non-cellulosic polysaccharides, especially heteroxylans and (1,3;1,4)-β-D-glucans, constitute a relatively high proportion of the walls of the aleurone and starchy endosperm, and probably also of the scutellum. In these tissues, cellulose contents are correspondingly lower. The generally low cellulose content of these walls, together with the fact that they contain no lignin, are thought to be related to a limited requirement for structural rigidity of walls in central regions of the grain, and to a requirement to rapidly depolymerize wall components following germination of the grain.
In contrast, in the cell walls of the pericarp-seed coat, which provides a protective coat for the embryo and endosperm and which is not mobilized during germination, cellulose and lignin contents are much higher and the concentrations of non-cellulosic polysaccharides are correspondingly lower.
(1,3;1,4)-β-D-glucans, also referred to as mixed-linkage or cereal β-glucans, are non-cellulosic polysaccharides which naturally occur in plants of the monocotyledon family Poaceae, to which the cereals and grasses belong, and in related families of the order Poales.
These non-cellulosic polysaccharides are important constituents of the walls of the starchy endosperm and aleurone cells of most cereal grains, where they can account for up to 70%-90% by weight of the walls.
Barley, oat and rye grains are rich sources of (1,3;1,4)-β-D-glucan, whereas wheat, rice and maize have lower concentations of this polysaccharide. The (1,3;1,4)-β-D-glucans are also relatively minor components of walls in vegetative tissues of cereals and grasses. Although present as a relatively minor component in vegetative tissues (1,3;1,4)-β-D-glucan) is still important in terms of, for example, the digestibility of vegetative tissue by animals and in the use of crop residues for bioethanol production.
(1,3;1,4)-β-D-glucans are important in large-scale food processing activities that include brewing and stockfeed manufacture. Moreover, the non-starchy polysaccharides of cereals, such as (1,3;1,4)-β-D-glucans, have attracted renewed interest in recent years because of their potentially beneficial effects in human nutrition.
However, despite this interest, major gaps remain in our knowledge of the genes and enzymes that control non-cellulosic polysaccharide biosynthesis, including (1,3;1,4)-β-D-glucan biosynthesis, in cereal grain.
(1,3;1,4)-β-D-glucan concentrations in grain are thought to be influenced by both genotype and environment. For example, the concentration of (1,3;1,4)-β-D-glucan in cereal grains depends on the genotype, the position of the grain on the spike and environmental factors such as planting location, climatic conditions during development and soil nitrogen.
However, the genes that contribute to (1,3;1,4)-β-D-glucan content in grain have not yet been identified.
The identification of genes encoding (1,3;1,4)-β-D-glucan synthases through traditional biochemical approaches has been seriously hampered by an inability to purify the enzymes to homogeneity. (1,3;1,4)-β-D-glucan synthases are membrane-bound and, therefore, are difficult to solubilise in an active form. In addition, (1,3;1,4)-β-D-glucan synthases rapidly lose activity following disruption of cells, and are likely to be present at very low abundance in the cell. Despite numerous attempts, purification of (1,3;1,4)-β-D-glucan synthases to homogeneity has not been achieved and, as a result, there are no reports of amino acid sequences obtained from the enzymes themselves. The inability to obtain even partial amino acid sequences from the purified (1,3;1,4)-β-D-glucan synthase enzyme has also prevented the identification and isolation of genes encoding (1,3;1,4)-β-D-glucan synthases.
However, identification of the genes encoding (1,3;1,4)-β-D-glucan synthases would be desirable, as this would facilitate modulation of the level of (1,3;1,4)-β-D-glucan produced by a cell, and therefore, allow the qualities of grain or vegetative tissue to be altered. Therefore, in order to enable the modulation of the level of (1,3;1,4)-β-D-glucan in a cell and associated changes in grain or vegetative tissue quality, there is a clear need to identify genes that encode (1,3;1,4)-β-D-glucan synthases.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.